Polyhexamethylene guanidine phosphate (PHMG) is an antimicrobial biocide that causes severe lung injury accompanied with inflammation and subsequent fibrosis. Cytokines mediate the inflammatory response, leading to fibrosis in injured tissues. PHMG is known to induce the expression of various cytokines in vitro and in vivo. In the present study, we investigated the involvement of three MAPK subfamilies (JNK, p38 MAPK, and ERK) in PHMG-induced cytokine expression in A549 human lung epithelial cells. Our in vivo and in vitro data indicated that PHMG induced an increase in mRNA expression of IL-6 and TNF-α, and enhanced the phosphorylation of JNK, p38 MAPK, and ERK. Further, we investigated the involvement of MAPKs in PHMG-induced mRNA expression of IL-6 and TNF-α using JNK, p38 MAPK, and ERK inhibitors in A549 cells. Pre-treatment with the JNK inhibitor but not the p38 MAPK or ERK inhibitor, significantly attenuated the PHMG-induced mRNA expression of IL-6 and TNF-α. These results suggest that the activation of JNK is involved at least partially in the induction of IL-6 or TNF-α expression by PHMG in A549 cells.